Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.     

Hypertonic saline dextran after burn injury decreases inflammatory cytokine responses to subsequent pneumonia-related sepsis

The present study examined the hypothesis that hypertonic saline dextran (HSD), given after an initial insult, attenuates exaggerated inflammation that occurs with a second insult. Adult rats (n = 15 per group) were divided into groups 1 (sham burn), 2 [40% total body surface area burn + 4 ml/kg isotonic saline (IS) + 4 ml.kg(-1).% burn(-1) lactated Ringer solution (LR)], and 3 (burn + 4 ml/kg HSD + LR), all studied 24 h after burns. Groups 4 (sham burn), 5 (burn + IS + LR), and 6 (burns + HSD + LR) received intratracheal (IT) vehicle 7 days after burns; groups 7 (burn + IS + LR) and 8 (burn + HSD + LR) received IT Streptococcus pneumoniae (4 x 10(6) colony-forming units) 7 days after burn. Groups 4-8 were studied 8 days after burn and 24 h after IT septic challenge. When compared with sham burn, contractile defects occurred 24 h after burn in IS-treated but not HSD-treated burns. Cardiac inflammatory responses (pg/ml TNF-alpha) were evident with IS (170 +/- 10) but not HSD (45 +/- 5) treatment vs. sham treatment (80 +/- 15). Pneumonia-related sepsis 8 days after IS-treated burns (group 7) exacerbated TNF-alpha responses/contractile dysfunction vs. IS-treated burns in the absence of sepsis (P < 0.05). Sepsis that occurred after HSD-treated burns (group 8) had less myocyte TNF-alpha secretion/better contractile function than IS-treated burns given septic challenge (group 7, P < 0.05). We conclude that an initial burn injury exacerbates myocardial inflammation/dysfunction occurring with a second insult; giving HSD after the initial insult attenuates myocardial inflammation/dysfunction associated with a second hit, suggesting that HSD reduces postinjury risk for infectious complications.     

Molecular and pharmacological approaches to inhibiting nitric oxide after burn trauma

Whereas controversial, several studies have suggested that nitric oxide (NO) alters cardiac contractility via cGMP, peroxynitrite, or poly(ADP ribose) synthetase (PARS) activation. This study determined whether burn-related upregulation of myocardial inducible NO synthase (iNOS) and NO generation contributes to burn-mediated cardiac contractile dysfunction. Mice homozygous null for the iNOS gene (iNOS knockouts) were obtained from Jackson Laboratory. iNOS knockouts (KO) as well as wild-type mice were given a cutaneous burn over 40% of the total body surface area by the application of brass probes (1 x 2 x 0.3 cm) heated to 100 degrees C to the animals' sides and back for 5 s (iNOS/KO burn and wild-type burn). Additional groups of iNOS KO and wild-type mice served as appropriate sham burn groups (iNOS/KO sham and wild-type sham). Cardiac function was assessed 24 h postburn by perfusing hearts (n = 7-10 mice/group). Burn trauma in wild-type mice impaired cardiac function as indicated by the lower left ventricular pressure (LVP, 67 +/- 2 mmHg) compared with that measured in wild-type shams (94 +/- 2 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dtmax, 1,620 +/- 94 vs. 2,240 +/- 58 mmHg/s, P < 0.001), and a lower rate of LVP fall (-dP/dtmax, 1,200 +/- 84 vs. 1,800 +/- 42 mmHg/s, P < 0.001). Ventricular function curves confirmed significant contractile dysfunction after burn trauma in wild-type mice. Burn trauma in iNOS KO mice produced fewer cardiac derangements compared with those observed in wild-type burns (LVP: 78 +/- 5 mmHg; +dP/dt: 1,889 +/- 160 mmHg/s; -dP/dt: 1,480 +/- 154 mmHg/s). The use of a pharmacological approach to inhibit iNOS (aminoguanidine, given ip) in additional wild-type shams and burns confirmed the iNOS KO data. Whereas the absence of iNOS attenuated burn-mediated cardiac contractile dysfunction, these experiments did not determine the contribution of cardiac-derived NO versus NO generated by immune cells. However, our data indicate a role for NO in cardiac dysfunction after major trauma.     

Hyperglycemia Exacerbates Burn-Induced Liver Inflammation via Noncanonical Nuclear Factor-κB Pathway Activation

Hyperglycemia and inflammation are hallmarks of burn injury. In this study, we used a rat model of hyperglycemia and burn injury to investigate the effects of hyperglycemia on inflammatory responses in the liver. Hyperglycemia was induced in male Sprague-Dawley rats with streptozotocin (STZ) (35-40 mg/kg), followed by a 60% third-degree scald burn injury. Cytokine levels (by multiplex, in cytosolic liver extracts), hormones (by enzyme-linked immunosorbent assay [ELISA], in serum), nuclear factor (NF)-κB protein deoxyribonucleic acid (DNA) binding (by ELISA, in nuclear liver extracts) and liver functional panel (using VetScan, in serum) were measured at different time points up to 7 d after burn injury. Blood glucose significantly increased after burn injury in both groups with different temporal patterns. Hyperglycemic rats were capable of endogenous insulin secretion, which was enhanced significantly versus controls 12 h after burn injury. DNA binding data of liver nuclear extracts showed a robust and significant activation of the noncanonical NF-κB pathway in the hyperglycemic versus control burn animals, including increased NF-κB-inducing kinase expression (p < 0.05). Liver acute-phase proteins and cytokine expression were increased, whereas secretion of constitutive proteins was decreased after burn injury in hyperglycemic versus control animals (p < 0.05). These results indicate that burn injury to the skin rapidly activated canonical and noncanonical NF-κB pathways in the liver. Robust activation of the NF-κB noncanonical pathway was associated with increased expression of inflammatory markers and acute-phase proteins, and impaired glucose metabolism. Hyperglycemia is detrimental to burn outcome by augmenting inflammation mediated by hepatic noncanonical NF-κB pathway activation.     

Graft survival and effectiveness of dermal substitution in burns and reconstructive surgery in a one-stage grafting model

Survival of the autograft and objective parameters for scar elasticity were evaluated after dermal substitution for acute burns and reconstructive surgery. The dermal substitute, which was based on bovine type I collagen and elastin-hydrolysate, was evaluated by intraindividual comparison in a clinical trial. The substitute was applied in a one-step procedure in combination with a split-thickness autograft. This treatment was compared with the conventional treatment, the split-thickness antograft. After 1 week, the percentage of autograft survival was assessed. The Cutometer SEM 474 was used to obtain objective measurements of skin elasticity parameters 3 to 4 months postoperatively. Forty-two pairs of wounds (31 patients, age 32.9 +/- 19.3 years; burned surface area, 19.8 +/- 14.5 percent) were treated because of acute burns. Reconstructive surgery was performed on 44 pairs of wounds (31 patients, age 33.9 +/- 17.5 years). Autograft survival was not altered by the substitute for reconstructive wounds, although a slight but significant reduction (p = 0.015) was established in the burn category for substituted compared with nonsubstituted wounds. However, the necessity for regrafting was not increased by substitution. Cutometer measurements of reconstructive wounds with a dermal substitute demonstrated a significant increase of pliability (50 percent, p < 0.001), elasticity (defined as immediate extension, 33 percent, p = 0.04), maximal extension (33 percent, p = 0.002), and immediate retraction (31 percent, p = 0.01), as compared with nonsubstituted wounds. After burn surgery, no improvement was found for the different elasticity parameters. Dermal substitution in a one-stage grafting model seems feasible with respect to graft survival. Skin elasticity was considerably improved by the collagen/elastin dermal substitute after reconstructive surgery.     

Comparison of donor-site healing under Xeroform and Jelonet dressings: unexpected findings

Split-thickness skin grafts remain central to the strategy of burn wound treatment. The dressing used to cover the donor wound site has a significant effect on healing parameters. The purpose of this study was to compare split-thickness skin graft donor site reepithelialization under Xeroform and Jelonet dressings. A dermatome was used to cut two consecutive strips of skin from 25 paired donor sites on the thigh, calf, or back of 19 participants. Standardization of the harvest method was achieved by using the same surgeon to harvest the compared skin graft strips, with attention to consistency of dermatome skin-thickness setting, downward pressure, and angle of dermatome approach. A strip of Xeroform or Jelonet was applied to one of each pair of wounds. Epidermal and dermal thickness was measured from biopsy specimens cut at the midpoint of each split-thickness graft strip. The day of final dressing separation was declared the day of complete donor reepithelialization (healing). The mean healing time for Xeroform and Jelonet was 10.4 +/- 2.6 days (n = 25) and 10.6 +/- 2.8 days (n = 25) (p = 0.76) at sites cut to a mean depth of 0.23 +/- 0.08 mm and 0.23 +/- 0.09 mm (p = 0.89), respectively. There was no correlation between graft thickness and healing time for sites dressed with Xeroform (r = 0.17) or Jelonet (r = 0.02). Donors sites reharvested 10 to 21 days after a prior harvest healed an average of 3.1 days earlier than virgin sites (8.4 +/- 1.6 versus 11.5 +/- 2.6 days, p < 0.001), although reharvested grafts were on average 0.05 mm thicker (p = 0.10). The mean thickness of reepithelialized donor-site epidermis (0.13 +/- 0.04 mm, n = 30) was found to be twice the thickness of virgin epidermis from the same sites (0.06 +/- 0.02 mm, n = 38, p < 0.001). Thirty-six grafts harvested with dermatomes set to cut 8/1000 inch (0.20 mm) deep ranged from 0.12 to 0.42 mm thick, with only eight of these grafts measuring within +/-10 percent of the desired thickness setting. Before donor dressing separation, Xeroform and Jelonet dressings were judged to be more comfortable by nine patients and one patient, respectively, whereas no difference was detected by six patients. The authors now use Xeroform as the preferred donor dressing.     

Effects of burn wound excision on bacterial colonization and invasion

Rates of survival after thermal injury have improved in the past two decades, and rates of wound infections and sepsis have decreased during the same period. Early excision has been advocated as one of the major factors, but its safety and efficacy and the exact timing of burn excision are still under debate. It was hypothesized that acute burn wound excision (in the first 24 hours after burning) would be superior to conservative treatment and delayed excision in preventing bacterial colonization and invasion. Twenty consecutive patients with thermal injuries were studied. Twelve patients underwent acute burn wound excision, and eight patients underwent conservative treatment and delayed excision. The second group of patients received topical treatments in another facility and underwent delayed excision after transfer to our service, on postburn day 6. Quantitative bacteriological assessments of the excised wound and biopsy samples of the wound bed, obtained before autografting and/or homografting, were performed. The effects of time on bacterial counts, differences between superficial and deep biopsy samples, and the effects of early versus late debridement were studied. Patients admitted early exhibited bacterial counts of less than 10 bacteria per gram of tissue. Patients in this group did not experience infection or graft loss. Patients admitted late exhibited counts of more than 10 bacteria (p = 0.001, compared with early admission). Three patients in the late excision group experienced infection and graft loss (p < 0.05, compared with the early excision group). Burn wound excision significantly decreased bacterial colonization for all patients (p < 0.001). Greater bacterial colonization and higher rates of infection were correlated with topical treatment and late excision (p < 0.001). It is concluded that burn wound excision significantly reduces bacterial colonization. Patients who undergo topical treatment and delayed burn wound excision exhibit greater bacterial colonization and increased rates of infection. Acute burn wound excision should be considered for all full-thickness burns.     

Lymphocyte DNA damage in patients with acute coronary syndrome and its relationship with severity of acute coronary syndrome

OBJECTIVE: The aim of this study was to investigate the association between lymphocyte DNA damage and acute coronary syndromes (ACS). METHODS: The study population contained 53 patients with ACS, 48 patients with stable angina and 35 voluntary healty subjects. DNA damage was assessed by alkaline comed assay in peripheral lymphocyte and plasma levels of total antioxidant capacity (TAC) were determined using a novel automated measurement method. RESULTS: In ACS patients, DNA damage was significantly higher than in patients with stable angina and control subjects (144+/-52 AU, 116+/-37, 68+/-34 AU; for three p<0.001, respectively). The TAC levels in patients with ACS were lower than the other groups (1.24+/-0.31 mmol Trolox equiv./l, 1.46+/-0.29 mmol Trolox equiv./l, p<0.05, respectively). DNA damage values in patients with acute miyocardial infarction were significantly higher than in patients with unstable angina (159.8+/-53.0 AU versus 131.8+/-48.4 AU; p<0.05, respectively). Lymphocyte DNA damage values in patients with ACS showed positive correlation with d-dimer (r=0.880, p<0.001) troponin I (r=538, p<0.001) and C-reactive protein (r=0.544, p<0.001) and negative correlation with TAC (r=-0.346, p=0.011). In multiple linear regression analysis, TAC (beta=-0.213, p=0.001) and d-dimer (beta=0.697, p<0.001) were independent predictors of DNA damage in patients with ACS. CONCLUSIONS:These findings indicate that lymphocyte DNA damage level increases in patients with ACS. Elevated DNA damage may be related with plaque instability and be useful for the identification of patients with acute coronary syndromes.     

Dura mater biology: autocrine and paracrine effects of fibroblast growth factor 2

The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells.Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.     

Towards quantitative assessment of burn based on photoacoustic and optical coherence tomography

Accurate and timely assessment of the severity of burn is essential for the treatment of burns. Currently, although most first‐degree and third‐degree burns are easily diagnosed through visual inspection or auxiliary diagnostic methods, the second‐degree burn is still difficult to distinguish due to the ambiguity boundaries of second‐degree with first‐degree and third‐degree burns. In this study, we proposed a non‐invasive technique by combing photoacoustic imaging (PAI) and optical coherence tomography (OCT) to multi‐parameter quantitatively assess the burns. The feasibility and capacity of the dual‐mode PAT/OCT for assessing the burns was first testified by tissue‐mimicking phantom and burn wounds in mouse pinna in vivo. The further experiments conducted on the back of rats showed that the changes in skin scattering structure, vascular morphology and blood flow provided by the dual‐mode PAI/OCT system can determine distinct boundaries and depth of the burns. The experimental results prove that combined PAI/OCT as a novel method can be used to assess the severity of burn, which has the potential to diagnose the burns in clinic.     

Timing of microcirculatory injury from ischemia reperfusion

The low flow state that results from ischemia and reperfusion injury is a potentially reversible process that is important in numerous clinical situations. However, the point in time during the course of reperfusion where tissue injury becomes irreversible is unknown. This experiment evaluated the continuum of tissue damage in skeletal muscle after ischemic insult by quantifying the number of flowing capillaries and percentage muscle necrosis in a male Wistar rat skeletal muscle model. A gracilis muscle flap was raised on the vascular pedicle of 39 male Wistar rats and examined at 832x using intravital videomicroscopy. The numbers of flowing capillaries in five consecutive high-power fields were counted for baseline values. The flap was then subjected to 4 hours of global ischemia (except in sham animals, n = 7) by placing a microvascular clamp on the pedicle artery and vein. Upon reperfusion, flowing capillaries were counted in the same five high-power fields at intervals of 5, 15, 30, and 60 minutes, then at 2 to 8 (1-hour intervals), 24, and 48 hours. The gracilis muscle was then harvested at these intervals during reperfusion and assessed for viability. Compared with baseline, flowing capillaries from the ischemia and reperfusion group (mean +/- SEM) decreased significantly in the first 8 hours of reperfusion (7.7 +/- 0.2 to 3.2 +/- 0.3, p < 0.001) with minimal change noted from 8 to 48 hours. Percentage muscle necrosis increased progressively in ischemia and reperfusion preparations from 1 to 7 hours of reperfusion (16.5 +/- 2.6 percent to 38.9 +/- 1.2 percent, p < 0.001). No significant change in muscle necrosis in the ischemia and reperfusion group was noted between 7 and 48 hours. Sham preparations showed no change in the number of flowing capillaries through 3 hours of reperfusion, with a slight decrease at 24 hours. This rat gracilis microcirculation skeletal muscle model demonstrates a heterogeneous reperfusion injury. The decrease in flowing capillaries correlated with the increase in percentage necrosis and appeared to stabilize at the 7- to 8-hour interval. This finding may have important implications for the timing of interventions aimed at minimizing tissue damage from ischemia-reperfusion.     

Observation of microvasculature in intestinal wall after burn in rats

OBJECTIVE: To investigate the relationship between intestinal mucosa injury and changes in stereologic parameters for its vessels. STUDY DESIGN: PAS stain was used to observe the basement membrane of intestinal epithelium. To measure the parameters of microvasculature by stereology, microvasculature was shown by alkaline phosphatase stain. Diamine oxidase (DAO) activity was determined by spectrophotometry. RESULTS: Red mucus became thinner than that of the control group; basement membrane was disrupted at 3 post burn hours (PBH). Changes became more serious at 6 and 12 PBH. Villus height and crypt depth appeared shortened at post burn. Microvasculature diameter was reduced. Mean section area at 12 PBH was significantly lower than that of the control group (p < 0.01). Mean circumference at 6 PBH was 19.43 +/- 1.59 microm and in the control group 23.84 +/- 4.17 microm. After burn injury, DAO activity in the intestine was reduced, falling to minimum at 12 PBH (p < 0.01), and in plasma was raised significantly, reaching peak at 12 PBH (p < 0.05). CONCLUSION: Our results suggest disruption of basement membrane, reduction of microvasculature diameter and DAO activity in intestine will appear at post burn, which induces decreased blood flow in intestine and damage to intestinal mucosa.     

Correlation between serum alkaline phosphatase activity and blood glucose levels

Fasting blood glucose levels and serum alkaline phosphatase activity of age-matched Libyan diabetic men (168) and women (168) were determined. The mean levels of blood glucose of men and women were 227 +/- 6 and 237 +/- 5 mg/dl, respectively. The respective values of serum alkaline phosphatase were 179 +/- 5 and 199 +/- 6 IU/l. The mean serum phosphatase activity of women was significantly higher (p less than 0.001) than that of their male counterparts. A statistically significant positive correlation was found between serum alkaline phosphatase and blood glucose levels of these diabetic patients (r = 0.35; p less than 0.001).     

Dynamic versus static splinting of simple zone V and zone VI extensor tendon repairs: a prospective, randomized, controlled study

The authors present the first prospective, randomized, controlled study comparing postoperative dynamic versus static splinting outcomes of patients following extensor tendon repair. Patients who incurred simple and complete lacerations of their extensor tendons in zones V and VI were enrolled into the study and underwent either static splinting (n = 17) or dynamic splinting (n = 17) following primary acute repair of tendons. Total active motion was improved in the dynamic group when compared with the static group in the injured digits at 4 weeks (180.5 +/- 4 degrees versus 131.3 +/- 61 degrees; p = 0.006), at 6 weeks (239 +/- 21.9 degrees versus 205.5 +/- 53.4 degrees; p = 0.048), and at 8 weeks (247+/- 19.8 degrees versus 216.3 +/- 36 degrees; p = 0.051), but not at 6 months (253.1 +/-18.8 degrees versus 250.5 +/- 32 degrees; p = 0.562). Similarly, total active motion averaged for all digits (injured and noninjured) of the involved hand was improved in the dynamic group over the static group at 4 weeks (209.8 +/- 31.3 degrees versus 140 +/- 58.2 degrees; p < 0.001) and at 6 weeks (241.5 +/- 17.2 degrees versus 217.1 +/- 42.4 degrees; p = 0.024), but not at 8 weeks (249.6 +/- 16 degrees versus 234.8 +/- 24.5 degrees; p = 0.215) or 6 months (252.3 +/- 14 degrees versus 249.1 +/- 31 degrees; p = 0.450). Grip strength outcomes demonstrated improved grip force for the dynamic group when compared with the static group at 8 weeks (81.3 +/- 18.0 percent versus 59.2 +/- 20.4 percent; p = 0.004) but not at 6 months (89.6 +/- 5.6 percent versus 82.1 +/- 22.0 percent; p = 0.595). Patients demonstrated forceful grip greater than or equal to 80 percent of the noninjured hand in 55 percent of patients in the dynamic group versus 15 percent of patients in the static group at 8 weeks. Patients demonstrated forceful grip greater than or equal to 80 percent of the noninjured hand in 100 percent of patients in the dynamic group versus 73 percent of patients in the static group at 6 months. The authors' findings suggest that dynamic splinting of simple, complete lacerations of the extensor tendons in zones V and VI provides improved functional outcomes at 4, 6, and 8 weeks but not by 6 months when compared with static splinting. Therefore, they recommend dynamic splinting of simple, complete extensor tendon lacerations in zones V and VI only to select patients who are motivated and desire earlier return to full functional capacity.     

Prognostic significance of erythroblasts in burns

Changes in hematopoiesis that occur in humans after a burn injury may have important effects on morbidity and mortality. In patients with a variety of severe diseases, the presence of erythroblasts in peripheral blood is known to be indicative of a poor prognosis. However, the prognostic significance of erythroblasts in peripheral blood of burn patients has not yet been estimated. This study included 464 consecutive burn patients, of whom 81 did not survive their injuries (17.5 percent). Together with erythroblasts in blood, data on age, sex, total burn surface area, third-degree burn, inhalation trauma, white blood cell count, C-reactive protein, and hemoglobin were studied. The mortality rate of patients with erythroblasts in peripheral blood (n = 53) amounted to 56.6 percent (n = 30; total burn surface area, 39 percent), which is significantly higher (p < 0.001) than the mortality rate of patients without erythroblasts (12.4 percent, n = 51; total burn surface area, 18.69 percent). None of the 10 patients with more than 1000 erythroblasts x 10/liter survived. The detection of erythroblasts in the peripheral blood of burn patients is highly predictive of death, with the odds ratio after adjustment for the other known prognostic factors being 8.3 (95 percent confidence interval, 4.5 to 15.3). Erythroblasts were detected for the first time on average 10 +/- 4 days (median, 6 days) after admission and 13 +/- 6 days (median, 7 days) before death. Detection of erythroblasts in burn patients is of high prognostic power with regard to in-hospital mortality, providing physicians with a strong prognostic method with which to identify seriously threatened patients. It seems attractive to think about an incorporation of erythroblasts into further refinements of burn scores.     

Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3

The water and solute transporting properties of the epidermis have been proposed to be important determinants of skin moisture content and barrier properties. The water/small solute-transporting protein aquaporin-3 (AQP3) was found by immunofluorescence and immunogold electron microscopy to be expressed at the plasma membrane of epidermal keratinocytes in mouse skin. We studied the role of AQP3 in stratum corneum (SC) hydration by comparative measurements in wild-type and AQP3 null mice generated in a hairless SKH1 genetic background. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries but were polyuric because of defective urinary concentrating ability. AQP3 deletion resulted in a > 4-fold reduced osmotic water permeability and > 2-fold reduced glycerol permeability in epidermis. Epidermal, dermal, and SC thickness and morphology were not grossly affected by AQP3 deletion. Surface conductance measurements showed remarkably reduced SC water content in AQP3 null mice in the hairless genetic background (165 +/- 10 versus 269 +/- 12 microsiemens (microS), p < 0.001), as well as in a CD1 genetic background (209 +/- 21 versus 469 +/- 11 microS). Reduced SC hydration was seen from 3 days after birth. SC hydration in hairless wild-type and AQP3 null mice was reduced to comparable levels (90-100 microS) after a 24-h exposure to a dry atmosphere, but the difference was increased when surface evaporation was prevented by occlusion or exposure to a humidified atmosphere (179 +/- 13 versus 441 +/- 34 microS). Conductance measurements after serial tape stripping suggested reduced water content throughout the SC in AQP3 null mice. Water sorption-desorption experiments indicated reduced water holding capacity in the SC of AQP3 null mice. The impaired skin hydration in AQP3 null mice provides the first functional evidence for the involvement of AQP3 in skin physiology. Modulation of AQP3 expression or function may thus alter epidermal moisture content and water loss in skin diseases.     

Tumour necrosis factor alpha, lipid peroxidation and NO* are increased and associated with decreased free-radical scavenging enzymes in patients with Weill-Marchesani syndrome

AIM: Weill-Marchesani syndrome (WMS) is a rare systemic disorder with both autosomal recessive and dominant inheritances. Accumulation of reactive oxygen species such as O2*-, H2O2 and OH* causes lipid peroxidation (LPO), whereas antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSHPx)) mediate defence against oxidative stress. Excess tumour necrosis factor (TNF)-alpha and NO* react with O2*- and cause further antioxidant depletion with an increase in mutation frequency by H2O2. This study investigated the levels of SOD, GSHPx, catalase (CAT), TNF-alpha, NO and LPO in patients with WMS. METHODS: A group of 10 WMS patients (four males, six females; age, 26.5+/-19.0 years) and 10 age-matched and sex-matched controls (five males, five females; age, 27.3+/-18.2 years) were included. Serum TNF-alpha levels were determined by a spectrophotometer technique using immulite chemiluminescent immunometric assay. Malondialdehyde (MDA) was determined in plasma; CAT in red blood cells (RBCs), and SOD and GSHPx in both plasma and RBCs. Total serum NO* levels were evaluated by Griess reaction. RESULTS: Mean levels of TNF-alpha (8.3+/-0.6 pg/ml) in WMS patients were significantly (p<0.001) higher than controls (4.3+/-0.2 pg/ml). Plasma MDA levels in patients and controls were 5.4+/-0.8 and 1.8+/-0.6 micromol/l, respectively, and the difference was significant (p=0.0002). SOD and GSHPx activities were significantly lower in both RBCs and plasma of WMS than in controls (RBC-SOD, 3981.9+/-626.6 versus 5261.6+/-523.0 U/g haemoglobin (Hb), p=0.0005; plasma-SOD, 529.4+/-49.3 versus 713.4+/-55.7 U/g protein, p=0.0002; RBC-GSHPx, 682.7+/-42.0 versus 756.5+/-47.6 U/g Hb, p=0.0011; plasma-GSHPx, 107.3+/-15.0 versus 131.4+/-19.7 U/g protein, p=0.0113). In addition, serum NO (NO*-2 + NO*-3) levels were also significantly (p = 0.0002) increased in WMS patients (54.4+/-5.7 versus 26.9+/-6.7 micromol/l). RBC-CAT levels were similar between groups (125.6+/-21.3 versus 131.0+/-21.5 k/g Hb, p = 0.8798). CONCLUSIONS: The elevated LPO, TNF-alpha and NO* with decreased antioxidant enzyme activities indicated impaired antioxidative defence mechanisms with an oxidative injury and cell toxicity in WMS patients. The use of multiple antioxidants and free radical scavengers might be helpful in this genetic disorder.     

Neuronal intracellular pH directly mediates nitric oxide-induced programmed cell death.

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH(i)) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 microM), 3-morpholinosydnonimine (300 microM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex anamine (300 microM). The pH(i) was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH(i) 7.36 +/- 0.01 to approximately 7.00 (p <.0001). Within 45 min, a biphasic alkaline response was evident, with pH(i) reaching 7.40 +/- 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pH(i) of 7.09 +/- 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 +/- 2% (24 h) and DNA fragmentation increased to 58 +/- 8% (24 h) (p <.0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH(i) of 7.60 +/- 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration.     

Leukotoxin,a linoleate epoxide: Its implication in the late death of patients with extensive burns

Burn death based on circulatory shock is often encountered after recovery from primary shock in patients with deep and extensive burns,i.e., late death. Several toxic substances have been proposed, however, the responsible substance remains obscure. Since we have found leukotoxin, a highly cytotoxic linoleate epoxide biosynthesized by neutrophils, in the burned skin, in the present study we determined plasma leukotoxin concentrations in various degree of 30 burn patients. C-reactive protein and circulatory white blood cells were also measured. A significantly high mortality rate of patients with extensive burns (burn surface area over 70%) was observed compared with that in patients with burn surface area under 70%, and significantly high leukotoxin concentrations were observed within a week, and 3 weeks after the thermal injury in patients with extensive burns compared with those in patients with burn surface area under 70%. There were two peaks of plasma leukotoxin concentrations,i.e., the early phase (within 1 week) and the late phase (over 1 week) in patients with extensive burns. Plasma leukotoxin concentrations significantly correlated with burn surface area in the early phase, and similar correlations were observed in the late phase. A significantly high mortality rate (61%) of patients with peak leukotoxin concentrations over 30 nmol/ml was observed compared with 8% for those below 30 nmol/ml. Plasma leukotoxin concentration correlated significantly to C-reactive protein concentration, log (leukotoxin nmol/ml)=0.042×C-reactive protein (mg/dl)+0.74, (r=0.83,P<0.01) in the late phase. From these results, it is concluded that leukotoxin is produced in patients with burns particularly in the late phase of extensive burns, and leukotoxin might play an important role in the tissue destructive procedure associated with severe burns.     

Ghrelin gastrokinetic action in patients with neurogenic gastroparesis

Ghrelin has been shown to accelerate gastric emptying in animals where its effect appeared mediated through the vagus nerve. We aimed to verify the gastrokinetic capacity of ghrelin in human. Patients with gastroparesis attributed to a neural dysregulation by diabetes (n = 5) or surgical vagotomy (n = 1) were evaluated. The emptying of a test meal (420 kcal) was determined by the C13 octanoic acid breath test. Saline or synthetic ghrelin 1-4 microg/kg were given in 1 min bolus at the end of the meal. T-lag and T-1/2 were shorter during ghrelin than during saline administration [33 +/- 5 min versus 65 +/- 14 min (p < 0.01) and 119 +/- 6 min versus 173 +/- 38 min (p < 0.001)]. Ghrelin injection therefore accelerated gastric emptying of a meal in humans even in presence of a deficient gastric innervation.